FIG 4.

NS1 C terminus of pandemic H1N1 inhibits ASC speck formation. (A) HEK293T cells were transfected with Myc-tagged NLRP3 or Myc-vector and Flag-tagged ASC or Flag-vector for 24 h and left uninfected (top three rows). The cells were transfected with Myc-tagged NLRP3 and Flag-tagged ASC for 12 h and left uninfected or infected with Hf09 or Hf09-817 at an MOI of 5 for 12 h (bottom three rows). The cells were fixed, permeabilized, blocked, and probed with appropriate antibodies, followed by DAPI staining. NLRP3-Myc (gray), Flag-ASC (green), viral NP (red), and nuclei (blue) were visualized by confocal microscopy. Scale bar, 10 μm. (B) ASC speck-forming cells and NP-stained cells infected by Hf09 or Hf09-817 were quantified from five randomly selected fields, and the percentages of ASC speck- and NP-positive cells in total NP-positive cells were shown. Results are representative of three independent experiments (**, P = 0.0025, Mann-Whitney test).